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NICEATM ICCVAM
NICEATM
ICCVAM
National Toxicology Program Interagency
Center for the Evaluation of Alternative
Toxicological Methods
Interagency Coordinating Committee on
the Validation of Alternative Methods
Report and Recommendations from
the NICEATM - ICCVAM International
Workshop on Alternative Methods for
Human and Veterinary Rabies
Vaccine Testing
Jodie Kulpa-Eddy, DVM, USDA
Richard McFarland, MD, PhD, FDA
Co-Chairs, ICCVAM Biologics Working Group
SACATM Meeting
September 6, 2012
National Institute of Environmental Health Sciences
Research Triangle Park, NC
2008-2013 NICEATM-ICCVAM Five Year Plan
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Vaccine Potency and Safety Testing
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One of ICCVAM’s highest priorities
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Multiple agencies involved
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Human vaccines: FDA, NIH-NIAID,
DOD, DHS, BARDA, CDC
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Veterinary vaccines: USDA, DOI,
DHS, NIH (Zoonotic diseases)
“NICEATM and ICCVAM will:
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Evaluate alternative test methods and
testing strategies for vaccine potency
testing
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Facilitate acceptance of adequately
validated test methods and humane
endpoints found to be sufficiently accurate
and reliable.”
http://iccvam.niehs.nih.gov/docs/5yearplan.htm
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Advance Alternative Test Methods and Strategies:
Vaccines and Biologics

Rationale for Priority
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
Vaccines/biologics accounts for majority of animals used for
testing (2/3)
Many animals experience significant unrelieved pain and
distress
Public Health Significance
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Biologics include vaccines, blood and blood components,
tissues, antibodies, and other substances used to treat or
protect against disease in humans and animals
http://iccvam.niehs.nih.gov/docs/5yrPlan/NICEATM5YR-Final.pdf
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
Animals Used in Testing that Involves Unrelieved
Pain and Distress (No Pain Relievers)
Animals by Testing Type
Reported to USDA (2010):
Biologics and
Vaccine
Testing
57%
(54,889)
• 57% (54,889) of the animals reported
to USDA that experience unrelieved
pain and distress are used for testing
Biologics and Vaccines
Toxicity and
Efficacy
Testing
38%
(37,108)
• Estimated that 2 million animals used
for testing that involves unrelieved
pain and distress (U.S.)
• Est. 95% of these are rats, mice,
and birds (not reported to USDA)
Research
5%
(5,126)
Data for all states with all animal data for Column E of APHIS Form 7023; USDA. 2010. Annual Report - Animal Usage by Fiscal Year. United
States Department of Agriculture. Animal and Plant Health Inspection Service. Available at:
http://www.aphis.usda.gov/animal_welfare/efoia/7023.shtml
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Based on NICEATM review of Column E justifications posted by USDA.
NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
Animals Used for Testing by Major Categories
(EU 2010)
Production and QC of
Medicines,
Biologics, vaccines etc.
Toxicity Testing
63%
(1,788,000)
37%
(1,044,000)
Total EU
annual animal
use for testing:
2,832,000
Majority of Animal Use is for Testing Biologics and Vaccines
Report on the Statistics on the Number of Animals used for Experimental and other Scientific Purposes in the Member
States of the European Union COM(2010) 511
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
NICEATM-ICCVAM International Workshop:
Human and Veterinary Vaccine Potency and Safety Testing

September 14-16, 2010 – Co-organizers
included:
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
Workshop Output:
-
http://www.sciencedirect.com/science/article/pii/S1877282X11000245
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EURL ECVAM, JaCVAM, Health Canada
Nearly 200 scientists, 13 countries
NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
Procedia in Vaccinology 5: 1-266 (2011)
Recommendations for the implementation
and use of alternative methods
Priorities identified for future work needed
to advance alternatives
Enhanced International harmonization
Conduct vaccine-specific workshop for
priority areas:
• Rabies (2011)
• Leptospirosis (2012)
• Pertussis vaccines (acellular) (2012)
• Diphtheria and tetanus toxoids (2013planned)
ICCVAM
NICEATM
NICEATM-ICCVAM International Workshop on
Human and Veterinary Rabies Vaccine Testing


October 2011: USDA Center for
Veterinary Biologics Ames, IA
Attended by over 80 human and
veterinary rabies vaccine experts
representing:
-
14 countries
Industry and academia
Regulatory authorities and
international organizations
•
FDA, USDA, NIAID, WHO, OIE,
PEI (Germany), IVI
(Switzerland), NVAL (Japan),
AHVL (UK) and National
Institute for Health Quality
Control (Brazil)
Rabies Workshop summary and discussion highlights are available at:
http://iccvam.niehs.nih.gov/meetings/RabiesVaccWksp-2011/RabiesVaccWksp.htm
ICCVAM
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NICEATM
Acknowledgements:
ICCVAM Interagency Biologics Working Group
U.S. Food and Drug Administration
Center for Biologics Evaluation and Research
•
Richard McFarland, PhD, MD (Co-Chair)
Center for Drug Evaluation and Research
•
Abigail Jacobs, PhD (CDER)
Center for Food Safety and Nutrition
•
Suzanne Fitzpatrick, PhD, DABT (CFSAN)
U.S. Department of Agriculture
Jodie Kulpa-Eddy, DVM (Co-Chair)
David Dusek, PhD
Donna Gatewood, DVM
Geetha Srinivas, DVM, PhD
Centers for Disease Control and Prevention
Susan Maslanka, PhD
Department of Defense
Department of Interior
Tonie Rocke, PhD
National Institute of Allergy and
Infectious Diseases/NIH
Suman Mukhopadhyay, PhD
National Institute of Environmental
Health Sciences/ NIH
William Stokes, DVM, DACLAM
Warren Casey, Ph. DABT
Health Canada Liaisons
Richard Isbrucker, PhD
Michele Regimbald-Krnel, PhD
EURL ECVAM Liaison
Marlies Halder, DVM
JaCVAM Liaison
Hajime Kojima, PhD
Leonard Smith, PhD
Janet Skerry
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Acknowledgements:
Organizing Committee
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
BWG Members
Subject Experts
William Stokes, DVM, DACLAM
NIEHS, USA
Warren Casey, PhD, DABT
NIEHS, USA
Jodie Kulpa-Eddy, DVM (Co-Chair)
USDA, USA
Richard McFarland, MD, Ph.D. (Co-Chair)
FDA, USA
Suman Mukhopadhyay, PhD
NIAID, USA
Geetha Srinivas, DVM, PhD
USDA, USA
Marlies Halder, DVM
EURL ECVAM, Italy
Richard Isbrucker, PhD
Health Canada, Canada
Hajime Kojima, PhD
JaCVAM, Japan
Michele Regimbald-Krnel, PhD
Health Canada, Canada
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
Donna Gatewood, DVM
USDA, USA
Robin Levis, PhD
U.S. FDA, USA
Charles Lewis, DVM
USDA, USA
Joseph Hermann, PhD
USDA, USA
Carmen Jungback, PhD
Paul-Ehrlich-Institut, Germany
Catherine Milne, PhD
EDQM, UK
Gayle Pulle, PhD
Health Canada, Canada
Mutsuyo Takayama-Ito, DVM, PhD
National Institute of Infectious Diseases, Japan
Maria Luz Pombo, PhD
PAHO, USA
Karen Brown, PhD
Pair O Doc’s Consultants, USA
Alexander Gaydamaka, DVM, PhD
ICCVAM
Merial/AHI, USA
NICEATM
Acknowledgements:
Workshop Organizers and Co-Sponsors


Organizers:
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National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM)
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Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM)
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European Union Reference Laboratory for Alternatives to Animal Testing
(EURL ECVAM)
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Japanese Center for the Validation of Alternative Methods (JaCVAM)
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Health Canada
Co-Sponsors
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Center for Veterinary Biologics, USDA
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International Alliance for Biological Standardization (IABS)
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National Institute of Environmental Health Sciences (NIEHS)
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National Toxicology Program (NTP)
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Society of Toxicology (SOT)
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Rabies Workshop Objectives
 Review state of the science of available
alternative methods; identify data gaps
 Develop implementation strategy to achieve
regulatory acceptance, implementation, and use
 Evaluate available replacement in vitro assays
for potency testing and process control
parameters and assays for lot consistency
 Evaluate potential reduction and refinements to
current in vivo test, where and when animal
testing still necessary
Rabies virus graphic utmb.edu
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
Rabies Workshop Program

Opening Plenary Session:
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Non-Animal Methods and Strategies
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Currently Available In Vivo Assays
Development and Validation of a Serological Method
Breakout Discussion Sessions:
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Antigen Quantification
New Technologies
Consistency of Manufacturing Parameters
Current NIH Research on Improved Rabies Vaccines
Reduction and Refinement Opportunities:
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Public Health and Animal Health Perspectives
US and International Regulatory Requirements
Industry Perspectives
In Vitro Antigen Quantification Methods
Antibody Quantification (Serologic Methods)
Refinement and Reduction Opportunities
NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
Workshop Rationale:
Rabies Vaccine Potency Testing
 Public health and animal
health1
-

15M people receive postexposure rabies prophylaxis
treatment each year
70,000 human fatalities annually
Dogs are dominant reservoir
outside developed countries
Regulatory requirements
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Global use of mouse vaccinationchallenge test
50,000-70,000 mice used per year
in US and EU for potency testing
and release of human and
veterinary rabies vaccines2
1Bouhry
H, Dautry-Varsat A, Hotez PJ, Salomon J. 2010. Rabies, still neglected after 125 years of vaccinations.
PLOS Neglect Trop D 4(11):e839.
2Bruckner
L, Cussler K, Halder M, Barrat J, Castle P, Duchow K, et al. 2003. Three Rs approaches in the quality
control of inactivated rabies vaccines. ECVAM Workshop Report 48. ATLA 31:429-454.
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
Current In Vivo Potency Method

NIH Challenge Test
- Immunization of mice with multiple dilutions of vaccine
- Intracerebral (IC) challenge ~14 days post
immunization
- Observed for 5-14 days post-challenge

Two sources of pain and distress
- IC challenge
• Some laboratories use general anesthesia without any
interference in test outcome
- Inadequately protected mice demonstrate clinical signs
of rabies (i.e. paralysis, paresis and convulsions) 6-9
days following challenge
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Current Status: Replacement (1)
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
The rabies virus spike glycoprotein (G protein) is the primary rabies
virus antigen shown to induce rabies virus neutralizing antibodies
The natively folded G protein is virion-associated, trimeric and highly
immunogenic.
In vitro potency tests must be able to distinguish between the highly
immunogenic (virion-associated, trimeric) and poorly immunogenic
forms.
Schematic diagram of a rabies virus particle.
http://www.cdc.gov/rabies/transmission/virus.html)
ICCVAM
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NICEATM
Current Status: Replacement (2)

Japan has developed and used for 10 years an ELISA for potency
release testing of a non-adjuvanted veterinary rabies vaccine.
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Monoclonal antibody utilized which detects only the conformationally
intact G protein.
A similar test is used in many laboratories to quantitate the G
protein content of in-process samples but not yet used as a
potency release test for other products.
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Workshop Recommendations: Replacement (1)
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Non-adjuvanted human and veterinary rabies
vaccines should be high priority for in vitro method
product specific validation
Manufacturers are encouraged to develop, validate,
and implement in vitro antigen quantification methods
to replace the NIH test
NICEATM-ICCVAM developed a comprehensive list
of available monoclonal antibodies for product specific
testing and validation
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Refinement and Reduction
Current Status: Serum Neutralization Test (SNT)
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Method used to detect and quantify antibodies in serum that
neutralize Rabies virus
SNT addresses Reduction and Refinement 3Rs goals by:
-
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Reducing animal use per test vaccine by up to 10-fold
Avoids pain and distress of IC challenge test
Reduced cost and more time effective
European Directorate for the Quality of Medicines and HealthCare
(EDQM) international collaborative study of 13 laboratories from 10
countries, including the US (USDA)1
- Confirmed accuracy and interlaboratory transferability of SNT
- SNT able to distinguish lots identified as sub-potent in IC
challenge test
- Group of Experts 15V recommended SNT for inclusion in
revised Ph. Eur. Monograph 0451 (Approved April 2012)
1Kramer
rabies
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B, Bruckner l, Daas A, Milne C. 2010. Collaborative study for validation of a serological potency assay for
vaccine (inactivated) for veterinary use. Parmeur Biol Scientific Notes 2: 37-55.
NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
SNT: Sufficiently Standardized for Product Specific
Validation (2)
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Based on results of interlaboratory validation study and
acceptance of the method in the Ph. Eur. Monograph
0451 for inactivated veterinary rabies vaccines, the SNT
is considered sufficiently standardized to provide an
alternative to the NIH challenge test.
Therefore, veterinary rabies vaccine manufacturers in
collaboration with regulatory authorities should:
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Initiate product specific validation with SNT
Validation should assess ability to identify subpotent lots
Validation of the multi-dilution SNT should progress
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To evaluate stability of test and reference vaccine lots
To calibrate new standards
To evaluate changes in the manufacturing process
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Workshop Recommendations: Reduction (1)
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Manufacturers and regulatory authorities
should investigate reduction in number of
mice used per dilution
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Especially at higher and lower vaccine
dilutions, and for vehicle, and positive control
groups
Human rabies vaccine manufacturers
should review historical test data for
justification to eliminate duplicate mouse
potency testing
Manufacturers should test multiple lots at
the same time
Regulatory authorities should establish
criteria that could avoid duplicate potency
testing upon vaccine importation
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Workshop Recommendations: Refinement (1)
 Regulatory agencies and global organizations should encourage:

Immediate incorporation of humane endpoints

Routine use of general anaesthesia for intracerebral injections
•
PEI Manuscript accepted in Biologicals (2012) – supported use
of anesthesia for rabies potency testing
 Provision of analgesics to avoid or minimize post-procedural pain
and distress
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Recommendations contingent upon studies to determine no
interference with the study outcome
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Investigate use of SR analgesics to avoid or minimize pain and
distress without interfering with study objectives
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Post-Workshop Implementation Actions for
Veterinary Vaccines: Refinement (2)

May 25, 2012: USDA CVB Notice 12-12: “Use of Humane
Endpoints and Methods in Animal Testing of Biological Products”
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CVB policy describing humane endpoints to be used in animal
challenge tests and use of anesthesia before and during intracerebral
(IC) challenge
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Animals exhibiting paresis, signs of paralysis, and/or convulsions must be humanely
euthanized and considered as deaths
•
Firms strongly encouraged to use anesthesia for IC challenge of mice
•
Encourages use of analgesics in animal studies and potency testing, when
no effect shown on study outcome
USDA CVB Notice 12-12:
http://www.aphis.usda.gov/animal_health/vet_biologics/publications/notice_12_12.pdf
ICCVAM
NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
NICEATM-ICCVAM International Workshop on
Human and Veterinary Rabies Vaccine Potency Testing
Summarizes invited speaker presentations, breakout group discussions and provides
detailed recommendations for the validation, implementation and use of alternative
methods.
Detailed appendices include:
•
•
•
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Best Practices for the Rabies Challenge Test
Serology Assay for Potency Testing of Inactivated Rabies Vaccines for Veterinary Use
Rabies Virus Monoclonal Antibodies for Potency Testing Inactivated Rabies Vaccines
Method of Production and Testing of Inactivated Veterinary Rabies Vaccines in Japan
Workshop report available online 10 August 2012:
http://dx.doi.org/10.1016/j.biologicals.2012.07.005
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
ICCVAM
NICEATM
Workshop Summary - Rabies Vaccine Potency Testing (1)
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Until in vitro replacements to the “NIH Challenge Test” are
validated and implemented, the following are encouraged:
Refinement
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Anesthesia: should always be used for IC injections
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Analgesics: should be provided to all mice; studies needed to
ensure no interference with test objectives
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Earlier humane endpoints: should be immediately incorporated in all
testing regulations where not already included
Reduction
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Evaluate potential for fewer dilutions, fewer mice per dilution, and
possible deletion of duplicate testing on each lot
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
Workshop Summary - Rabies Vaccine Potency Testing (2)
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Replacement of the “NIH Challenge Test”
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
Serological Methods: Serum Virus Neutralization Test (SNT)
•
Eliminates the need for challenge with live rabies virus: complete
refinement
•
Sufficiently standardized per recent int’l study; manufacturers should
conduct and submit product-specific validation studies, in consultation
with regulatory agencies
Complete animal replacement
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In vitro antigen quantification methods:
•
Product specific validation should proceed with suitable monoclonal
antibodies; must discriminate sub-potent lots
•
Requires monoclonal antibodies specific for the trimeric glycoprotein G
ICCVAM
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NICEATM-ICCVAM - Advancing Public Health and Animal Welfare
NICEATM
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